HUMAN PROTOPORPHYRINOGEN OXIDASE - EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CLONED ENZYME

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependen t oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherich ia coli, purified to homogeneity, and characterized. Northern blot ana lysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is app roximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these c ells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homod imer composed of subunits of a molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extincti on coefficient of 48,000 at 270 nm and contains no detectable redox ac tive metals. The apparent K-m and K-cat for protoporphyrinogen IX are 1.7 mu M and 10.5 min(-1), respectively. The enzyme does not use copro porphyrinogen III as a substrate and is inhibited by micromolar concen trations of the herbicide acifluorfen. Protein database searches revea l significant homology between protoporphyrinogen oxidase and monoamin e oxidase.